The present invention concerns a system for producing multiple diagnostic test elements comprising,
a support with an analytical region for applying diagnostic test spots and one or several stop edges for each direction in space to position the support,
a first holding unit into which the support is inserted and positioned by means of one or several stop edges,
a first printing head which is arranged above the holding unit and which is used to release fluid drops of a first liquid onto the analytical region of the support,
a first positioning unit which laterally displaces and positions the first holding unit,
a second holding unit into which the support is inserted and positioned by means of one or several stop edges,
a second printing head which is arranged above the second holding unit and is used to release fluid drops of a second liquid onto the analytical region of the support,
a second positioning unit which laterally displaces and positions the second holding unit,
a transport unit which transports the support into the holding units,
a control unit which controls the transport unit, the holding units and the positioning units and the release of liquid from the printing heads.
The present invention additionally concerns multiple diagnostic test elements in which a plurality of test spots are arranged particularly exactly in a predetermined pattern and also a production process for such test elements and an analytical method using multiple test elements.
Test elements have already been known for some time in the prior art in which various reagents are applied to a support. For example an indicator is described in the German Patent DE 27 27 347 in which two or several substances are applied to zones on the support surface on a support which have a certain spacing. Printing techniques such as screen printing or electrostatic printing and the so-called ink-jet printing are disclosed for applying the reagents to the surface. A process for the production of reagent supports is also described in U.S. Pat. No. 4,216,245 in which two or several reagents are applied in non-overlapping regions of the surface. In the aforementioned documents this procedure is chosen to avoid undesired interactions of the reagents. In contrast test elements are described in the U.S. Pat. Nos. 5,378,638 and 4,591,570 that are suitable for immunological tests. A method is described in U.S. Pat. No. 4,591,570 in which various solutions of antibodies are applied manually to the support using a pipette. The diameter of the surface regions (spots) is between 0.25 and 1 mm. An apparatus for the automatic production of supports containing a plurality of small test spots is described in U.S. Pat. No. 5,338,688. The support to be coated is located on a positioning table which can be moved laterally and a single printing head for applying drops of liquid is located above the support. In this document it is pointed out that it is possible to generate patterns of various types of liquid which are arranged alternately. However, it is not shown how such a pattern can be generated in practice.
The object of the present invention was to propose a device and a process for the production of multiple test elements which produces test elements that have particularly small test zones in a highly accurate predetermined arrangement. In particular an object of the present invention was to provide such precise test elements in which the test spots have different compositions. An additional object of the present invention was to propose a device for producing test elements and a suitable process that can be used to reliably and rapidly produce the test elements according to the invention.
The present invention has achieved the said objects by providing a system as claimed in claim 1. This system can be used to produce multiple test elements which have a very accurate pattern of small test zones.
Multiple test elements which can be produced with a production system of the present invention have a support on which an analytical region is located. A plurality of essentially circular test spots is applied in the analytical region in a specified pattern. The diameter of the individual test spots is preferably less than 350 xcexcm and the centres of the test spots deviate by less than 40 xcexcm from the specified pattern. Such an accurate arrangement of test spots is unknown in the prior art. Letters and such like can also be generated by a pattern of very accurately arranged individual spots when using ink-jet printing heads in conventional printers. However, a problem in the present invention is that the test zones of the multiple test elements cannot be applied with a single printing head and instead several printing heads filled with different printing liquids are required. Colour printers are known in the prior art in which different liquids are applied using several printing heads or one printing head with several chambers. In such colour printers the paper and the printing heads are moved. Such an arrangement is less suitable for the production of very precise test elements since neither the absolute nor the relative accuracies of the positioning are adequate. In addition colour printers of the prior art do not enable the high printing densities required for the present invention composed of different test spots that are separated from one another. It was found that the positioning inaccuracy is mainly due to the fact that the printing heads are moved during the printing process. Other disadvantages of colour printers are the use of a relative large distance between the discharge nozzle of the printing head and the printed material. The present invention discloses an improved method and system for producing exact test elements with different test spots which avoids the disadvantages of the prior art.
In the production of multiple test elements the manufacture already knows the desired pattern so that a deviation of the test spots from the pattern and the extent of the deviation can be simply determined by a microscopic evaluation or similar methods. The deviation from the pattern can, however, also be determined when the specified pattern is unknown since the patterns used in practice are strict geometric arrangements such as squares, rectangles, honeycombs etc. In the said patterns there are always rows of test spots. Hence, in order to determine deviations from the pattern, the centres of test spots that are located in a row can be joined together by a fitting line. The deviation from the pattern is given in a simple manner by the distance of the individual test spot centres from the line of fit. In this definition of distance it is assumed that different types of test spots are in the observed rows. It is technically relatively simple to achieve the inventive precision for rows of similar test spots which can be produced with one and the same printing head.
The analytical region of multiple test elements according to the invention is preferably planar and has an unevenness of less than 100 xcexcm, particularly preferably of less than 10 xcexcm. The analytical region is preferably made of a material into which the liquids cannot penetrate or only to a very slight extent. Suitable materials are in particular plastics of which polystyrene has proven to be particularly advantageous. However, the analytical region can also be composed of glasses, metals or silicon which have suitable surface properties. The analytical region onto which the test spots are applied is preferably hydrophobic in order to avoid bleeding of the printed liquid drops. The use of a hydrophobic surface for the analytical region, compatible printing liquids to generate test spots and suitable methods for applying test spots can ensure that the test spots have a defined size and shape.
The analytical region should also have a sufficiently homogeneous surface which has only a few defects and little unevenness. The defects on the surface should be so small that the longest dimension of the imperfection is less than 10 xcexcm and the unevenness of the surface should be preferably less than or equal to 10 xcexcm.
A preferred use according to the invention of the multiple test elements is for analyses using fluorescent light. In this case it has proved to be advantageous to select materials at least for the analytical region and preferably for the entire support which on the one hand are not excited by fluorescent radiation to emit light and on the other hand do not reflect the fluorescent radiation that is used. Such materials can be obtained in a suitable manner by mixing plastics with carbon black.
Multiple test elements can be obtained according to the invention which have the shape of a strip or such like and are dipped into a sample solution or onto which the sample solution is applied. However, it has turned out that the multiple test elements according to the invention can be used particularly well for detection methods in which the test spots are incubated together with the sample. These are in particular immunological test methods in which an antigen-antibody reaction takes place. In order to carry out such an incubation with very small amounts of sample, the analytical region of the multiple test elements is surrounded by a rim such that the rim and analytical region together form a well. The sample liquid to be examined and optionally additional liquids such as reagents are added to this well. Such a multiple test element enables a plurality of analytical tests to be carried out using sample volumes of a few microlitres. The height of the said rim is adapted to the amount of liquid which is to be held by the well. The rim typically has a height of about 2 mm.
It has turned out that it is advantageous for the production of a multiple test element according to the invention and for carrying out analytical tests if the support of the test element has a basal surface which is parallel to the analytical region and serves to position the support vertically. In particular it is advantageous when the basal surface used for positioning is arranged on the same side of the support as the analytical region. In addition it has turned out according to the invention that the positioning is particularly exact and the production process is particularly optimal when the support is inserted from the underside into a holding unit in which the test spots are applied. Therefore the region of the basal surface used as a stop is preferably on the surface of the support where the analytical region is also located.
Furthermore it is advantageous for the production and use of the multiple test elements when one or several cross-pieces are mounted on the basal surface which run essentially perpendicular to the basal surface and provide the stop edges for two directions in space. For manufacturing reasons it has proven to be advantageous when the cross-pieces are located on the underside of the basal surface i.e. on the side of the support which faces away from the analytical region. The cross-pieces serve to position the test elements in a plane (lateral positioning). It has proven to be advantageous when the test elements have pairs of cross-pieces which are opposite one another so that a lever for positioning can engage with one cross-piece of such a pair and the second cross-piece of the pair is pressed against a stop in a holding unit. In addition cross-pieces have proven to be advantageous which have a U shape in a top-view on the basal surface of the support. This type of cross-piece enables the multiple test elements according to the invention to be stacked and on the other hand they can be displaced horizontally in the stacked state if two U shaped cross-pieces are used with a spacing in between which is larger than the diameter of the well.
A system for producing multiple test elements has at least two holding units in which the supports or the multiple test elements can be inserted. The holding unit has several stop edges against which the stop edges of the supports are pressed. Usually three different types of stop edges are used. On the one hand the basal surface of the support is moved against one or several stop edges which position it in the vertical direction. The support is preferably inserted from below into the holding unit and the holding unit has stops against the underside of which the upper side of the support is positioned. In addition a holding unit has stop edges for the two lateral directions in space against which the support is positioned.
Printing heads are arranged above the holding units from which drops of liquid are applied to the analytical regions of the support. The printing heads for liquids described in U.S. Pat. No. 5,338,688 and EP-A-0 268 237 are for example suitable as printing heads. However, other printing heads known in the prior art are also suitable such as those used for ink-jet or bubble-jet printers. Since such printing heads are widely used a more detailed description is not given but rather reference is merely made to the complete contents of the already mentioned patent documents.
The invention requires that the printing heads can deliver defined amounts of liquid in the order of magnitude of 1000 pl or less in a reproducible manner. In this connection it is necessary that the volume can be kept constant between successive printing processes and this also applies to the speed with which the drops hit the analytical region. If the said parameters are kept constant, it can be ensured that the shape and size of the individual test spots is always the same and thus a regular pattern is obtained. These requirements can for example be met by a printer of the type ADK-201b from the Microdrop Company in Hamburg and a suitable electrical control (MD-E-201 from the same company). Another suitable printing head control is also described in EP-A-0 268 237. The printing heads which are parts of a production system are advantageously adjusted such that they deliver the same amount of liquid at the same speed. This also usually results in the same size of test spots for different printing liquids. If the viscosities or surface tensions of the individual printing liquids are very different it may be necessary to experimentally adjust the control of the printing heads such that test spots of equal size are obtained for different printing liquids.
In the production system the printing heads are firmly positioned and are not moved. It has turned out that this is of major importance for the accuracy and reproducibility of the production process. It has proven to be much more reliable to re-position a support to be printed under each of the printing heads rather than to move and thus to position the printing heads over the support. Although the printing heads of the production system according to the invention are not moved in order to deliver a drop of liquid, each of the printing heads can be adjusted to enable a correction of the lateral printing head position if it turns out that the test spots that are produced by a particular printing head deviate too strongly from the specified pattern.
Such an adjustment of the printing heads is carried out by a quality assurance device which is an optional component of the production system. A quality assurance device optically examines the multiple test elements and checks whether the test spots of an analytical region deviate from a specified pattern. Deviations from this pattern are converted into signals which carry out an adjustment of one or several printing heads. The optical examination of the test elements can be carried out by CCD cameras which monitor the maintenance of a specified pattern by means of image processing software. It is also possible to use printing liquids which contain one or several fluorescent dyes and to determine the position of the test spots by means of a fluorescence microscope or a fluorescence scanner.
It has proven to be advantageous for the production of multiple test elements according to the invention to adhere to certain process parameters for the printing. The fluid drops which emerge from the printing heads should have a speed of less than 5 m/s, preferably less than 2 m/s. It has been shown that these speeds are optimal for the production of uniform test spots and for avoiding splashing of the drop on the analytical surface. With the said speeds of the fluid drops it is advantageous to reduce the distance between the printing head nozzles and the analytical region to below 2 mm or even better below 500 xcexcm in order to make the printing process as independent as possible from interferences of the path of the drop during flight and to be able to use low flight speeds. The volume of the individual fluid drops which are applied to the analytical region is preferably less than 1000 pl in order to produce sufficiently small test spots. In addition it is advantageous when the analytical region is hydrophobic in order to avoid bleeding of the applied drops. The surface is preferably made in such a way that a critical angle of more than 70xc2x0 and even better of more than 80xc2x0 is obtained with a 20 mM potassium phosphate solution.
Due to the very small amounts of liquid that are used in the printing process, drying is very rapid under normal room conditions. However, this fact which at first appears to be favourable, has proven to be disadvantageous when immunological test elements are produced. It has turned out that the relatively large antigen or antibody molecules require an adequate time in order to align themselves in a suitable manner on the surface of the support. This is especially the case when an analytical region is used which is coated with an antigen or antibody and the printing liquid contains a corresponding binding partner to which the reagent (usually also an antigen or antibody) is bound. This embodiment of the invention has the advantage that the binding partner and thus also the reagent can be firmly bound to the surface. In order to allow an adequate period for alignment of the binding partner molecules, the printing process is preferably carried out in an environment with an elevated relative air humidity, preferably 80%-98%. The air humidity is controlled by sealing off the entire production system or parts of the system to the environment by means of a housing and setting a specified air humidity within this housing by deliberate evaporation or condensation of water.
A production system according to the invention additionally has one or several positioning units for each holding unit which laterally displace and position the respective holding unit. A support is positioned and held within the holding unit as described above. The positions within the analytical region on which a fluid drop is to be applied are selected by means of the positioning unit. For this the positioning unit is steered by a control unit such that the holding unit is moved laterally to a certain position. The positioning unit can for example contain threaded rods which move in threaded holes in the holding unit and are driven by stepping motors which in turn are controlled by the control unit.
Multiple test elements according to the invention can each contain one or several test spots of a particular reagent. The latter is advantageous to ensure the entire test element does not become useless if there is a failure of a single test spot. On the other hand it is advantageous to have several test spots of one type in order to be able to average the analytical results that are obtained which compensates for errors when carrying out the analysis or for manufacturing variations of the test spots.
If a multiple test element has several test spots of the same type, then these are preferably applied onto the test element by one and the same printing head. For this the support is inserted and positioned in a holding unit, the holding unit is moved into a first position by its positioning unit and a fluid drop is released by the respective printing head. Subsequently the holding unit is moved into a further position of the specified pattern and an additional fluid drop is delivered. The method used in this process in which a single drop or a few drops are released in a time interval after selective control of the printing head is referred to as drop on demand.
As already described above, an essential characteristic of the present production system is that it has several holding units into which one and the same support is successively inserted and printed with liquid drops. Therefore the support is transported with the transport unit into different holding units in order to produce a multiple test element.
A robotic arm is for example suitable as a transport unit which grips a fresh support, inserts it in a first holding unit, removes it after the printing process and inserts it into a second holding unit etc.. However, it has proven to be more advantageous to use a transport unit which comprises a transport device for a lateral transport as well as a lifting device for the vertical transport of the supports. The transport device is preferably a conveyer belt which runs below the holding units. Furthermore it is preferable not to use a common conveyer belt with a planar transport belt but rather a conveyer belt on which there are holders to hold supports. Holders are for example possible which are permanently connected to the conveyer belt. In addition holders can be used which have pins etc. on their underside which engage in corresponding recesses of the conveyer belt. The said holders preferably have a socket shape on which the supports are placed. If supports are used with cross-pieces on the underside, it is advantageous when these cross-pieces are then situated on the sides of the holder and thus prevent the support from sliding off the holders. The lifting device of the transport unit lifts the support from the conveyer belt into the holding unit. The production system preferably has one lifting device for each holding unit. Such a lifting device can for example be a push rod which is moved vertically upwards by means of a motor from a first position in which it is located below the support in the process of which it grips under the support and, together with the support, moves into a second position in which the support is pressed against the vertical stop of the holding unit. Hence it is possible to design the lifting device so simply that only one movement in a direction in space has to be carried out. In this connection it is advantageous when two opposite sides of the support protrude over the holder on which it is located. In such an arrangement the lifting device can be moved directly to the support from the underside.
A control unit controls the movement of the transport unit, the holding and positioning of the supports in the holding units, the movement of the positioning units as well as the release of liquid from the printing heads. A conventional microprocessor can for example be used as a control unit which is equipped with suitable interface cards. The control unit regulates the process for producing diagnostic multiple test elements based on a stored programme. Such a production process which is also a subject matter of this invention comprises the following steps:
Positioning a support in a first holding unit below a first printing head,
Moving the first holding unit and the support positioned therein with a positioning unit into a position in which a first site of the analytical region is positioned below the first printing head,
Release of one or several liquid drops from the first printing head onto the first site of the analytical region,
Positioning the support in a second holding unit below a second printing head,
Moving the second holding unit and the support positioned therein with a positioning unit into a position in which a second site of the analytical region is positioned below the second printing head,
Release of one or several liquid drops from the second printing head onto the second site of the analytical region,
wherein the support is moved into one or several additional positions while it is located in a holding unit in which drops of liquid are released to generate a specified pattern of test spots in the analytical region.
The process described above is so exact that a pattern of test spots can be generated in which the centres of the test spots deviate by less than 40 xcexcm from the specified pattern.
The control unit described above also controls the holding units in order to hold and position the supports in the holding units. Furthermore the control unit controls the transport unit in order to supply the holding units with supports in a suitable time sequence.
The present invention also concerns an analytical method using multiple test elements according to the invention comprising the steps:
applying a sample liquid to the analytical region of a multiple test element,
recording an image of the analytical region with the pattern of test spots arranged therein,
evaluating the regions of the image in which test spots are located,
determining the presence and/or the concentration of one or several analytes.
When test spots are used which change colour with an analyte, the multiple test element can be evaluated by a simple optical examination for example with a microscope or a CCD camera. However, according to the invention it is preferable to carry out analytical tests in which a fluorescent signal is formed or disappears when the test spots react with analytes. Such an analytical method can for example be carried out with a sandwich assay in which an antigen is located in a test spot which can be used to detect an antibody which may be present in the sample liquid. The antigen of the test spot reacts with the antibody from the analyte and forms a complex to which an additional antigen can bind which is labelled with a fluorescent dye. The labelled antigen can for example be derived from a reagent to which the sample liquid has previously been added or it can also be derived from a part of the surface of the support where it has previously been applied similarly to a test spot. The fluorescence signal generated in such a sandwich assay can also be recorded with a CCD camera that is sensitive for the emitted fluorescent light. The analytical region of a multiple test element can also be examined in a fluorescent microscope (see EP-A-0 601 714) or with a fluorescent scanner (see WO 96/02824). Images are obtained with the described methods which can either be evaluated visually or with the aid of a microprocessor.